Currently, international concern is heightened regarding the potential use of smallpox (variola) virus as a bioterrorism agent [1],[2]. This concern has increased since the tragic events around September 11th and the fall of 2001, particularly since the virus has been eradicated [3]. Thus, the recommendations developed by key advisory groups, such as the CDC Advisory Committee on Immunization Practices (ACIP) regarding vaccinia (smallpox) vaccine and the potential use of smallpox virus as a biological weapon (Modlin, 2001; see www.cdc.gov) are important guidance documents. Recommendations from the ACIP included the need to develop new vaccines, particularly a reformulated vaccine produced using cell culture techniques, and better research reagents and methods for therapy based on more modern technologies. Additional recommendations regarding vaccination of persons at various levels of risk, including acute or workplace exposure to infectious or highly attenuated strains, have been revised as part of those recommendations.
The degree to which the individuals who received the original smallpox vaccinations are protected is still a matter of conjecture and debate [3-7]. It is well known that long-lasting protective immune responses against vaccinia virus have been historically documented [3]. Cytotoxic T lymphocytes (CTLs; CD4+ and CD8+) are generated following immunization and memory T cells can be re-stimulated many years later by a variety of in vitro methods [6], and kinetics of antibody formation upon re-vaccination have been defined [8].
As reported by the ACIP and other sources (e.g., see www.cdc.gov), Dryvax,® the vaccinia (smallpox) vaccine currently licensed in the United States, is a lyophilized, live-virus preparation of infectious vaccinia virus (Wyeth Laboratories, Inc., Marietta, Pa.). The vaccinia vaccine does not contain smallpox (variola) virus. Previously, the vaccine had been prepared from calf lymph with a seed virus derived from the New York City Board of Health (NYCBOH, 9) strain of vaccinia virus and has a concentration of about 108 pock-forming units (PFU)/ml. Vaccine was administered by using the multiple-puncture technique with a bifurcated needle. Although generally not life-threatening, there are some side effects in a subset of immunized individuals, particularly those who are immunosuppressed. Current stockpiles of vaccine kept at the CDC are inadequate, even when only high-risk for exposure individuals (e.g., military personnel and “first responders” ) are targeted for potential immunization with dilute vaccine, but particularly so in the case of a national emergency. Because the vaccine technology for vaccine production and immunization against smallpox is very old and stockpiles are inadequate in light of potential bioterrorism with smallpox, there is a need for better vaccines, state-of-the art methods of large-scale vaccine production and safer methods for re-immunization as well as de novo immunization of individuals at risk or for immunization of the public in the case of a national emergency. Furthermore, even though recent work indicates that diluted stockpiled vaccine is still immunogenic, even at dilute doses (39,40), the use of the NYCBOH strain and bifurcated needle delivery techniques are fraught with the problems of a replicating virus that can ooze from pustules at the immunization site consequently posing a potential threat to immunocompromised individuals.
Vaccinia virus strains with changed virulence have been developed and provide useful vectors for gene therapy and other applications [10]. The most attenuated strain, MVA, has acquired multiple deletions and mutations through serial passage [11-14]. The virus can replicate in chick embryo fibroblasts, but it has a very limited mammalian host cell range. Human cells are non-permissive for virus replication, which is blocked in the late stage of infection. The recommended mammalian host cell is the baby hamster kidney 21 clone 13 (BHK21-CL13) cell line [15].
Many studies support the selection of MVA as a therapeutic or prophylactic vaccine in humans. The virus has been used effectively for primary vaccination against smallpox in a test of 120,000 recipient vaccines [11]. When used as a vector, MVA has proved to be an efficient system because it expresses high levels of heterologous microbial and tumor antigen genes [13-21] in the absence of viral replication [18, 21]. An added margin of safety has been demonstrated from use of this strain in animals and human clinical studies [22-26], including immunocompromised individuals [27-33]. Of particular relevance to oral delivery are studies of Belyakov et al. [34] demonstrating strong mucosal and systemic immunity following intrarectal administration of recombinant MVA in a mouse vaccine therapy model.
In addition to safety and efficacy, oral immunization with replication-deficient recombinant vaccinia virus such as MVA, offers many other advantages over other vaccine candidates. The method effectively induces immune response in all three arms of the immune system, i.e., serum antibody, mucosal IgA antibody, and cell-mediated immunity. Compliance may be increased and overall costs reduced, because use of an oral rather than a parenteral vaccine may enhance patient (and/or parental) acceptance and would obviate the need for syringes and needles. Recombinant MVA can be constructed for multivalent vaccine or as a cocktail of MVA vaccines. Lyophilized vaccinia is extremely heat-stable. Heating to 100° C. for two hours led to a loss of only one log of infectivity, and storage at 45° C. for 2 years was still 100% successful in vaccination of volunteers [35]. These properties make oral MVA an ideal candidate vaccine.
Large-scale production of MVA is envisioned as a safe and immediate vaccination approach. Validation of vaccine safety and efficacy by bioassay on human intestinal cells is one method in the scope of evaluation of MVA safety so that it meets pre-clinical and clinical testing standards for vaccine production. The human intestinal cells are nonpermissive for the virus, as are other human cells, but they produce viral antigens that are recognized by the host's immune effector cells to stimulate systemic and mucosal immunity. A further benefit to the use of the intestine-derived cells as part of the testing and validation regimen is that it is well recognized that these types of epithelial cells can also act as antigen presenting cells in vivo. Thus, this is an excellent approach to induce effective immunity.